Currently 6% of males, of fertile age, in western countries have some kind of disease which prevents normal reproduction. To this effect, the World Health Organisation (WHO) has put together a series of laboratory procedures in a single protocol which standardises the analysis of the quality of semen in the international setting. These studies are centred on the determination of the concentration, morphology and motility of the sperm, complemented with the evaluation of certain functional tests, as well as determined biochemical and enzymatic parameters of the semen (WHO, 1999). This group of tests can estimate the total volume of the same and the concentration of sperm per milliliter and it can be diagnosed whether the infertility of the male is due to an absence (azoospermia) or a clear decrease (oligospermia) by the quantity of sperm in the ejaculate. Also, it determines the possible existence of motility problems (asthenozoospermia) which makes it impossible for these cells to cross the uterine cavity and successfully reach the outer third of the Fallopian tubes. It is also analysed whether there are serious morphology problems of their components (head, neck, tail) (teratozoospermia), given that these variations have repercussions in the capacity for an efficient fertilisation of the female ovule. Additionally, it likewise explores the participation of the glands such as the prostate and seminal vesicles (infections, agenesis). Lastly, with functional tests such as the HOS test (ionic permeability of the cell membrane) or the progression capacity of the sperm in vitro, they give an idea of the fertility capacity of the semen. Finally, these laboratory studies occasionally have to be complemented with hormone profiles, testicle biopsy and/or the determination of the karyotype (chromosome study which defines the hereditary condition of the male or female sex of an individual) and/or molecular genetic tests.
Despite clinical and laboratory studies, the cause of the infertility cannot be determined in about 30%-50% of infertile males, being called idiopathic infertility. Recently, it has been recognised that damage of the sperm DNA could explain a high percentage of these cases (Evenson et al., 1999; Larson et al., 2000), in such a way that the study of DNA fragmentation of sperm is a subject of active research with continuous publications in specialised journals (Evenson et al., 2002). Chromatin anomalies, or even damage in the nuclear DNA of the sperm, could take place or even be the result of anomalies in the DNA packaging which takes place during spermatogenesis (Sailer et al., 1995). There is also even the possibility that they may be the result of damage produced by free radicals which cause oxidative stress (Aitken et al., 1998), a consequence of a possible apoptosis process (Gorczyca et al., 1993).
There are different methods for evaluating the integrity of the chromatin/DNA of human sperm. Among them is highlighted the rupture of DNA in situ by introducing labelled nucleotides in the same using enzymes such as terminal transferase (TUNEL) or DNA polymerase (in situ nick translation ISNT) (Gorczyca et al., 1993). These methods are based on the use of enzymes on the sperm fixed on slides. For that reason their efficiency is not very high, only those labelled breakages being accessible to the enzyme, which leads to a relatively low reproducibility of the results. The reagents are also expensive, therefore the techniques are only used in research, not being possible to use them for the clinical evaluation of semen. Another technique is the comet assay (Hughes et al., 1996). The sperm are included in an agarose microgel on a slide and are subjected to lysis solutions to extract the membranes and proteins. Thus, nucleoids are obtained, that is, deproteinised nuclei, in which the DNA loops have been unwound due to stretching. The nucleoids are subjected to electrophoresis in a tank filled with buffer, in such a way the DNA strands migrate to the anode, creating the image of a comet, with a head and a tail in the direction of the electrophoretic migration. These comets are stained with a fluorescent stain, to be observed under a fluorescence microscope. If the nucleus has DNA fragmentation, a large quantity of them will have migrated, and concentrated in the tail of the comet. It is a fairly sensitive test, but also relatively expensive and complicated for a conventional clinical laboratory. In fact, it requires particularly uncommon equipment: electrophoresis power source and tank, fluorescence microscope, and an image capturing system and the analysis of the same. For all these reasons it is not applicable to the clinical study of semen either and is only used for research purposes.
The current reference technique for the study of DNA fragmentation of sperm is the chromatin structure assay by Evenson (SCSA: Sperm Chromatin Structure Assay; Evenson et al., 1980; 2000; Evenson and Jost, 1994). In this technique, the sperm in suspension are added to an acid denaturing solution. Those sperm without breakages in their DNA are resistant to this acid denaturing, remaining as double strand DNA. However, the sperm with fragmented DNA themselves denature their DNA, being transformed into single chain DNA. They are then stained with acridine orange. This stain emits a green fluorescence when it binds with double stranded DNA. However, in the sperm with denatured DNA, in a single strand, this fluorochrome emits a red fluorescence. The sperm with denatured DNA are quantified using flow cytometry, to discriminate between both types of fluorescence. The SCSA is a technique with wide clinical scope, having been evaluated in a large number of patients. Using this system, it has been established that when an individual has 30% or more of the sperm with fragmented DNA, their probability of a pregnancy going to term is less than 1%, and this applies in natural fertilisation as well assisted production (Evenson et al., 1999; Larson et al., 2000).
The percentage of sperm with fragmented DNA can be more or less constant in the different spermatogenesis cycles of an individual, but can also vary due to exogenous factors, or, for example, after an intense febrile episode, such as influenza (Evenson et al., 2000). In this way, serial studies can be made, selecting those samples with a lower level of fragmentation, to subsequently be used in assisted production techniques. It is important to take into account that freezing the semen samples in liquid nitrogen does not alter the levels of DNA fragmentation, therefore this test can be done on frozen samples, which can later be used in insemination, IVF (in vitro fertilisation) or ICSI (Intracytoplasmatic Sperm Injection). This has a great operational advantage for the patient and the laboratory.
The SCSA technique, although robust and highly reproducible, is an expensive system, difficult to implement, and not very accessible to the routine laboratory (De Jonge, 2002). For this reason, the quality of the sperm DNA still cannot be evaluated routinely, despite its verified clinical value in the study of infertility.
Recently, our research group preliminarily described a technique which enabled the chromatin of human sperm to be dispersed in situ, demonstrating that those sperm incapable of dispersing the chromatin contained fragmented DNA (Fernandez, J. L. et al., Journal of Andrology, 2003, vol. 24, No. 1, pp. 59-66; “The Sperm Chromatin Dispersion Test: a simple method for the determination of sperm DNA fragmentation”). Using this method, samples of semen are treated sequentially in agarose microgel with an acid denaturing solution, with two lysis solutions and with a wash so that they may be dried and stained afterwards. This technique, which is called Sperm Chromatin Dispersion (SCD) test, uses excessively aggressive reagents and conditions. The described method does not give consistent results which makes repeated evaluations difficult. On the other hand, the quality and contrast of the images obtained and the reproducibility of the results are not good enough to be able to be applied commercially. Also, the structure of the sperm is affected and the tail is not visible in the samples. This problem is important, since the sperm cannot be easily distinguished from other cells in the sample, with the subsequent error in quantifying the number of sperm with damaged chromatin/DNA.
Therefore there is still the need of a reliable process which could be used routinely and easily for the study of the quality of semen in animals and in particular to evaluate the integrity of the chromatin/DNA. The process has to be robust, easy to implement, cheap and accessible to the basic laboratory. It has to resolve the previously mentioned problems. It also has to give homogenous results between laboratories and be suitable for automation.